Project Summary Gene expression is regulated by transcription factors, chromatin structure and higher order organization. The positioning of genes within the nucleus and with respect to each other often correlates with their expression. The molecular mechanisms that control gene positioning within the nucleus are largely unknown. As a model for gene positioning, we have focused on the molecular mechanisms by which genes reposition from the nucleoplasm to the nuclear periphery upon activation in Saccharomyces cerevisiae. Targeting to the nuclear periphery involves a physical interaction with the nuclear pore complex (NPC) and is mediated by cis-acting DNA zip codes that are both necessary and sufficient to induce interaction with the NPC and localization at the nuclear periphery. Such zip codes also confer interchromosomal clustering of genes at the nuclear periphery. We have identified a number of transcription factors that bind to these zip codes and mediate targeting to the nuclear periphery. This suggests that controlling gene positioning is an unappreciated function of transcription factors and that cis-acting transcription factor binding sites represent a genetic means to control the spatial organization of the genome. The proposed studies will 1) determine the molecular mechanism by which the Gcn4 TF mediates targeting to the nuclear periphery and interchromosomal clustering, 2) comprehensively identify TFs and DNA binding proteins that impact gene positioning, and 3) test the hypothesis that TFs spatially and functionally compartmentalize the yeast genome.